1. Field of the Invention
The invention relates to the identification of a circulating protein associated with extracellular fiber matrix metabolism in mammalian connective tissues. More specifically, it is directed to assays for the detection and quantitation of molecules and fragments of YKL-40, a protein associated with connective tissue metabolism in mammals. It also involves correlating serum levels of YKL-40 in a mammal to the presence and status of diseases in which matrix metabolism plays a role, such as joint disorders the metastasis of certain tumors.
2. Description of Related Art
The extracellular matrix of mammalian connective tissues (such as articular cartilage of joints and vascular wall tissue of the vascular and lymphatic system) provides strength to, and (to varying degrees) a barrier to the migration of cells from, the tissue. In certain disease processes, however, the matrix is degraded by hydrolytic enzymes. As the matrix degrades, the integrity of the tissue is impaired, which may allow tissue cells, by-products and other residues of the matrix metabolism to escape into bodily fluids and/or lymphatic or vascular circulation. Detection of these molecules and cells can, in certain instances, provide information regarding the biochemical characteristics of the extracellular matrix, including how it is synthesized and how it is lost. Also, where a particular molecule that is produced and/or secreted during abnormal matrix metabolism is closely related to a disease process, quantitation of that molecule in the patient""s body fluids and/or tissues can help clinicians monitor the progress of the disease.
Human joint cartilage is known to contain several different types of proteins and proteoglycans, a few of which are present only in cartilage. These matrix constituents are released from cartilage tissue as it degrades during the course of certain joint diseases. The quantity of released matrix constituents (including fragments thereof and related macromolecules) present in a particular fluid or tissue may correlate with the intensity of the disease. Conversely, where the damage to the cartilage is reversible (as in secondary reactive arthritis caused by infection of the joint tissue), a reduction in levels of previously measured released matrix constituents may correlate with the degree of remission of the disease.
In practice, however, identification of reliable markers for metabolism of cartilage and other connective tissues and development of assays for their detection has proved to be a difficult task. Certain released fragments and molecules may be rapidly cleared from circulation by the lymph nodes, liver and phagocytosis (see, e.g., Frazer, et al., Hyaluronan: Sources, Turnover and Metabolism, Clinical Impact of Bone and Connective Tissue Markers 31-49 (Acad. Press, 1989); Smedsrod, xe2x80x9cCatabolism in Liver Sinusoidsxe2x80x9d, id. at 51-73; and, Heinegard, et al., Brit. J. Rheumatol., 30 (Suppl. 1): 21-24, 1991). Further, certain molecules are present in several different connective tissues, thus making correlation to metabolism in a particular tissue based on circulating levels of the molecule uncertain. Even where levels of a particular molecule can be traced to metabolism in the tissue of interest, the molecules may decline to undetectable levels or be biochemically altered in structure during those stages of a disease when a substantial quantity or connective tissue has been lost.
Not surprisingly, therefore, attempts to develop assays, especially those utilizing serum, which correlate levels of certain proteins to joint disease activity have met with mixed success. Rohde and co-workers have described radio-immunoassays (RIAs) for serum levels of amino-terminal type III procollagen peptide and its degradation products in rheumatoid arthritis (RA) patients (Rhode, et al. Eur. J. Clin. Invest., 9:451-459, 1979). This propeptide (P-III-NP) can be detected in several body fluids; a subsequent report attempted to correlate serum levels of P-III-NP to disease activity using the Rhode, et al. radioimmunoassay (Hxc3x8rslev-Petersen, et al., Arth. and Rheum., 5:592-599, 1986). While the concentrations of serum P-III-NP were significantly elevated in patients with active RA, these concentrations were also elevated to a similar degree in patient""s with inactive RA, thus making the distinction between the two states based on P-III-NP levels alone difficult.
Assays of serum levels of other connective tissue metabolites and constituents in RA patients have been attempted in connection with treatment protocols to gauge the success of those protocols, again with mixed success. For example, Hxc3x8rslev-Petersen, et al., ibid, measured serum levels of P-III-NP, immunoreactive propyl 4-hydroxylase protein (1 RPH), 7S domain of collagen type IV (Col IV, 7S) and fragment PI of laminin (S-Lam), which are associated with metabolism of extracellular interstitial collagens and basement membranes. Although serum levels of P-III-NP, 1 RPH and Col IV, 7S were elevated in RA patients (as compared with healthy adults), the levels did not decline to normal even with apparent remission of the disease. Also, levels of S-Lam remained normal in both active and inactive RA patients. As a result, the presence and quantity of these proteins in serum does not appear to clearly correlate to the progress or remission of RA.
Similar difficulties have also prevented the identification of reliable markers for the progress of other connective tissue diseases. Identification of candidate molecules and fragments which may serve as reliable markers for connective tissue metabolism is, therefore, an important goal of clinical chemistry research. To this end, the expression of given proteins by matrix-forming cells has been assessed by immunologic assays for antigen and by hybridization assays for mRNA encoding candidate marker protein. Isolation of proteins from the extracellular matrix is, however, limited to the identification of secreted proteins that become abundant constituents of that matrix. As a result, identification of candidate proteins has been limited.
In 1992, the inventors described a method for identification of all proteins secreted by a matrix-forming cell (Johansen, et al., J. Bone and Min. Res., 7:501-512, 1992). Using this method, a 40 kD protein was identified as a secreted protein of human bone cells. The inventors hypothesized that the protein (named YKL-40 after the first three amino acids at the N-terminus and the molecular weight) could play a role in the action of Vitamin D in bone. YKL-40 appears to be the same protein identified by Rejman, et at., (Biochem. Biophys. Res. Commun., 150:329-334, 1988) as being present in mammary secretions of non-lactating cows whose mammary glands were undergoing involution.
As described in detail below, it has since been discovered that YKL-40 can serve as a reliable marker for joint disease, including diseases with disparate pathologies such as rheumatoid arthritis and osteoarthritis. Surprisingly, it has also been discovered that serum levels of YKL-40 are also substantially elevated in patients with metastasis of breast cancer cells, particularly those patients who survive for a relatively short period of time following recurrence and metastasis of their cancer. Further, the inventors have determined that significantly elevated levels of YKL-40 appear in the sera of persons having connective tissue degradation in organs such as the liver and prostate.
The methods for detecting and quantifying levels of YKL-40 in biological samples described herein, therefore, provide a means of charting the progress of not only joint disease, but also cancer cell metastasis. Further, based on the apparent relationship of serum levels of YKL-40 to connective tissue metabolism, it can be reasonably predicted that the methods described will be of use in the diagnosis and monitoring of other diseases in which connective tissue metabolism plays a role, such as osteoporosis.
Detection and quantitation of a marker for diseases whose activity can be correlated to loss and/or synthesis of connective tissue matrices can be of value in diagnosing and monitoring the progress of both the disease and its amelioration. One such marker is YKL-40, a protein of about 40 kD molecular weight which has been found in elevated concentrations in the blood and synovial fluid of human patients with joint disease, as well as in the blood of human patients with breast cancer or disorders of organs such as the liver.
A point of commonality between these conditions is their relationship to connective tissue loss. Specifically, connective tissue loss in joint disease results from its degradation by enzymes released in the disease process. In cancer cell metastasis, it is believed that degradation of the connective tissue of vessel walls and, possibly, of body organs permits migration of cells from the primary cancer tissue. Similarly, with the loss of organ tissue in an organ degenerative disease such as cirrhosis, it can be expected that connective tissue within the organ may also be degraded.
An object of the invention, therefore, is to provide a method of detecting and quantifying YKL-40 in biological samples using an antibody specific for YKL-40 and, where appropriate, a detectably labelled antigen (YKL-40).
Another object of the invention is to provide methods for diagnosis of diseases which are correlated to the loss and/or synthesis of connective tissue as indicated by levels of YKL-40 detected in a biological sample. In this respect, the invention is expected to be of particular use in the diagnosis of joint disease (such as RA), cancer cell metastasis (as in, for example, breast cancer), diseases related to loss of connective tissue in bone (such as osteoporosis and osteoarthritis), and diseases related to loss of connective and/or other tissue in organs (such as cirrhosis of the liver).
Another object of the invention is to provide methods for the quantitation of levels of YKL-40 to monitor the progress and/or amelioration of a disease which is associated with connective tissue metabolism related to the presence of YKL-40. Again, the invention is expected to be of particular use in tracking the progress and/or amelioration of joint disease (such as RA), of cancer cell metastasis (as in, for example, breast cancer), diseases related to the loss of connective tissue in bone such as osteoporosis and osteoarthritis, and diseases that result in the loss of connective tissue in organs, such as cirrhosis of the liver.